Membrane-type MMPs are indispensable for placental labyrinth formation and development. We have demonstrated that MT1-MMP deficiency is compatible with development, however, postnatal survival and growth is substantially reduced due to the loss of ability to turn over collagen matrices in connective tissues. These observations, while illustrating the importance of collagen remodeling, also highlights that MT-MMP function is redundant and we have embarked on a systematical dissection of the function of each MT-MMP with specific emphasis on MT1-MMP, MT2-MMP and MT3-MMP. From these studies, we have previously described that MT3-MMP works in concert with MT1-MMP in early development targeting similar substrates of the fibrillar collagen types I, II, and III. Combined loss of MT1-MMP and MT3-MMP leads to demise immediately following birth with severe skeletal defect and cleft palate in the majority of pups. All double deficient animals do however go to term and we therefore reasoned that another MT-MMP family member with a wider expression pattern could be responsible for the rescue, or that MT-MMP activity simply is dispensable prior to birth. To test this hypothesis, we generated mice that are MT2-MMP deficient under the assumption that MT2-MMP could compensate for the loss of MT1-MMP and MT3-MMP. Our results show that MT2-MMP mice are healthy and grossly indistinguishable from wild type littermates. To determine if the combined activity of MT-MMPs is required for development, we crossed the MT2-MMP deficient mouse strain to the MT1-MMP deficient mouse strain. Intercrossing of mice doubly heterozygous for the two MT-MMPs failed to produce offspring with combined MT1-MMP/MT2-MMP deficiency. To account for this apparent deficit in greater detail, we collected embryos from timed pregnant female mice and established that double deficient offspring were present at E9, but subsequently involuted between E10.5 and E11.5. Closer inspection of the embryos revealed signs of placental defects manifested by grossly dilated vessels. Accordingly, histology of the placentas from double deficient embryos displayed a significant defect in the investment of embryonic vessels and establishment of the labyrinth layer of the placenta. where exchange of nutrients and gasses take place between maternal blood sinuses and the embryonic vasculature. In the absence of gross deposits of matrix components around the rare stunted vessels found in mutant placentas, we pursued a role for matrix metalloproteinases in a different function. Notably, electron microscopy analysis of the placenta revealed a failure to establish the two-syncytiotrophoblast layers around the embryonic vessels, which are required for normal labyrinth formation. To address whether or not there was a constitutive requirement for MT1-MMP and MT2-MMP in development, we employed a conditional MT1-MMP allele developed in our laboratory. In conjunction with an inducible cre recombinase, the conditional allele facilitated deletion of MT1-MMP in an MT2-MMP deficient background. We eliminated MT1-MMP at predetermined times following administration of the inducing agent to pregnant female mice carrying embryos with the inducible deficiency for MT1-MMP and unconditional MT2-MMP deficiency. When MT1-MMP expression was eliminated in an MT2-MMP deficient background prior to the formation of the placental labyrinth, the embryos underwent involution with placental defects as observed with unconditional MT1/2-MMP deficiency. However ablation of MT1-MMP and MT2-MMP following the formation of the placental labyrinth was compatible with development to term. In addition, induction of double deficiency immediately following birth was compatible with postnatal growth and the animals were grossly indistinguishable from mice with single deficiency for MT1-MMP. Consistent with the observed expression of MT1-MMP and MT2-MMP in placenta, we conclude, that these proteinases singly and in concert, mediate proteolysis of substrates in the placenta essential for the branching of embryonic vessels. In particular, we note the absence of syncytiotrophoblast layers around the embryonic vessels. Differentiation of chorionic trophoblasts into syncytiotrophoblasts is driven by co-opted retroviral proteins (syncytins). We suggest based on our observations, that MT-MMP activity in the placenta affects the cell fusion processes required for this syncytia formation. Our observations constitute the first example of MMP activity required for development in the mouse and highlights the essential role these proteases play in early development.